Stem cells are like a treasure box containing numerous effective ingredients useful for stimulating stem cell growth/regeneration, repairing and/or rejuvenating damaged/aged tissues, treating degenerative diseases, and preventing tumor/cancer formation. Hence, it is conceivable that we can use these stem cells as a tool for novel drug identification and production. As a result, the drugs so obtained are useful for developing pharmaceutical and/or therapeutic applications, such as a biomedical utilization, device and/or apparatus for diagnosis, stem cell generation, stem cell research and/or therapy, tissue/organ repair and/or rejuvenation, wound healing treatment, tumor suppression, cancer therapy and/or prevention, disease treatment, drug production, and a combination thereof.
MicroRNA (miRNA) mir-302 is the most predominant RNA found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is largely unclear. Previous studies have shown that ectopic overexpression of mir-302 beyond the level found in hES cells is able to reprogram both human normal and cancerous cells to normal hES-like pluripotent stem cells with a slow cell cycle rate (20-24 hour/cycle) and dormant-cell-like morphology (Lin et al., 2008 and 2011; U.S. Ser. No. 12/792,413; EP2198025). Relative quiescence is a defined characteristic of these mir-302-induced pluripotent stem (mirPS) cells, whereas hES and other previously reported three/four-factor-induced (by Oct4-Sox2-Klf4-c-Myc or by Oct4-Sox2-Nanog-Lin28) iPS cells have all shown highly proliferative ability (12-15 hour/cycle) and inexorable tumorigenetic tendency (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007). Despite the unknown mechanism underlying this anti-proliferative characteristic of mirPS cells, we have identified the possible involvement of two mir-302-targeted G1-checkpoint regulators, cyclin-dependent kinase 2 (CDK2) and cyclin D (Lin et al., 2010; U.S. Ser. No. 12/792,413). Progression in the eukaryotic cell cycle is driven by activities of cyclin-dependent kinases (CDKs), which forms functional complexes with positive regulatory subunits, cyclins, as well as by negative regulators, CDK inhibitors (CKIs, such as p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1). In mammalian cells, different cyclin-CDK complexes are involved in regulating different cell cycle transitions, such as cyclin-D-CDK4/6 for G1 progression, cyclin-E-CDK2 for G1-S transition, cyclin-A-CDK2 for S-phase progression, and cyclinA/B-CDC2 (cyclin-A/B-CDK1) for entry into M-phase. Hence, it is conceivable that the anti-proliferative function of mir-302 likely results from such co-suppression of CDK2 and cyclin D during G1-S transition.
However, studies of the mir-291/294/295 family, an analog to human mir-302 in mouse, revealed a totally different function from the role of mir-302 in human cells. In mouse embryonic stem (mES) cells, ectopic expression of mir-291/294/295 promoted fast cell proliferation and G1-S transition through direct silencing of p21Cip1 (also named CDKN1A) and serine/threonine-protein kinase Lats2 (Wang et al., 2008), leading to high tumorigenecity of the transfected cells. It has been known that transgenic mice lacking p21Cip1/Waf1 were shown to display normal development with a defect in the G1 checkpoint control (Deng et al., 1995). Yet, the role of Lats2 remains to be determined because of its function in recruitment of gamma-tubulin and spindle formation at the onset of mitosis. Loss of Lats2 in mouse embryos was found to cause severe mitotic defects and lethality, indicating that silencing of Lats2 may hinder rather than facilitate cell division (Yabuta et al., 2007). Taken together, silencing of p21Cip1 seems to be the key mechanism underlying such mir-291/294/295-induced tumorigenecity. Nevertheless, our efforts to screen the mir-302 target site in human p21Cip1 gene all resulted in negative. The same negative result has also been shown by two of the most renowned online miRNA-target prediction programs TARGETSCAN (http://www.targetscan.org/) and PICTAR-VERT (http://pictar.mdc-berlin.de/). Therefore, although mir-302 and mir-291/294/295 are homologous analogs, their functions in regulating cell tumorigenecity are actually opposite to each other, indicating that homologous microRNAs may not possess the same function! This finding also suggests that current studies using siRNA mimics to replace natural microRNAs may not deliver the same result as well!
The genomic sequence encoding mir-302 is located in the 4q25 locus of human chromosome 4, a conserved region frequently associated with longevity. More precisely, mir-302 is encoded in the intron region of the La ribonucleoprotein domain family member 7 (LARP7) gene and expressed via an intronic microRNA biogenesis pathway (Barroso-delJesus, 2008; Ying and Lin, 2004; FIG. 13). Native mir-302 consists of four familial homologues sense (mir-302b, c, a and d) and three distinct antisense members (mir-302b*, c* and a*), all of which are transcribed together as a polycistronic RNA cluster along with another miRNA, mir-367 (Suh et al., 2004). In our priority invention U.S. Ser. No. 12/792,413, we have observed that mir-302 can stimulate the expression of other miRNAs, such as mir-92, mir-93, mir-367, mir-371, mir-372, mir-373, mir-374, and the whole mir-520 familial members. Analyses using the online “TARGETSCAN” and “PICTAR-VERT” programs in the Sanger miRBase::Sequences website (http://microrna.sanger.ac.uk/), further revealed that mir-302 shares over 400 target genes with these stimulated miRNAs, suggesting that they may also play a similar role like mir-302. These shared target genes include, but not limited, members of RAB/RAS-related oncogenes, ECT-related oncogenes, pleiomorphic adenoma genes, E2F transcription factors, cyclin D binding Myb-like transcription factors, HMG-box transcription factors, Sp3 transcription factors, transcription factor CP2-like proteins, NFkB activating protein genes, cyclin-dependent kinases (CDKs), MAPK/JNK-related kinases, SNF-related kinases, myosin light chain kinases, TNF-alpha-induce protein genes, DAZ-associated protein genes, LIM-associated homeobox genes, DEAD/H box protein genes, forkhead box protein genes, BMP regulators, Rho/Rac guanine nucleotide exchange factors, IGF receptors (IGFR), endothelin receptors, left-right determination factors (Lefty), cyclins, p53 inducible nuclear protein genes, RB-like 1, RB binding protein genes, Max-binding protein genes, c-MIR cellular modulator of immune recognition, Bcl2-like apoptosis facilitator, protocadherins, integrin β4/β8, inhibin, ankyrins, SENP1, NUFIP2, FGF9/19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, histone acetyltransferase MYST3, nuclear RNP H3, and many nuclear receptors and factors. The majority of these target genes are highly involved in embryonic development and cancer tumorigenecity. Thus, it is conceivable that miR-302 can further stimulate its homologous microRNAs, such as miR-92, miR-93, miR-367, miR-371, miR-372, miR-373, miR-374, and miR-520, to enhance and/or maintain its function.
MicroRNA (miRNA) is a cytoplasmic gene inhibitor and often functions to suppress the translation of it targeted gene transcripts (mRNAs) via binding to a specific target site with high complementarity and then forming a RNA-induced silencing complex (RISC) to block or degrade the mRNAs. Hence, the binding stringency between miRNA and its target genes determines the real function of a miRNA. To this, our previous studies (Lin et al., 2010; U.S. Ser. No. 12/792,413) have provided significant insights into the mechanism underlying mir-302-mediated tumor suppression. In humans, mir-302 targets multiple cell cycle regulators CDK2, cyclins D1/D2 and BMI-1 genes for silencing, but interestingly, not p21Cip1. As aforementioned, simultaneous silencing of CDK2 and cyclin D blocks the cell cycle transition from the G1 to S phase, leading to a slow cell proliferation rate. In addition, silencing of BMI-1 stimulates the elevation of two major tumor suppressors p16Ink4a and p14ARF expression, which may further attenuate cell proliferation and inhibit tumor formation. Since p16Ink4a/p14ARF are elevated while p21Cip1 may not be affected in human cells, the anti-tumorigenecity function of mir-302 likely results from both p16Ink4a-Rb and/or p14/19ARF-p53 pathways in addition to the co-suppression of cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways.
Based on the above anti-tumorigenecity feature of mir-302, we may use mir-302 as a drug to treat human tumors and cancers. However, there are four major problems in this attempt: (1) As a gene silencing effector, mir-302 must function through its precursor form, a hairpin-like 73-75-nucleotide RNA that still can not be made by any currently available RNA synthesis technology. To ensue its sequence fidelity, the maximal length of a synthesized RNA is around 45-55-nucleotide, which is not sufficient to form a mir-302 precursor (pre-mir-302). (2) We also need a high concentration of mir-302 beyond the level found in hES cells in order to induce its anti-tumor/cancer effects (Lin et al., 2010). Yet, there is currently no method capable of generating such a high amount of pre-mir-302 for affordable drug production. Isolating pre-mir-302 from hES or iPS cells is very costly, whereas bacterial competent cells are not able to produce the high secondary structure of a pre-mir-302. (3) Synthetic siRNA mimics have never succeeded in animal trials. Due to the short life (3-5 days) and high toxicity of the siRNA mimics, siRNA can not be used to replace the natural mir-302 under an in vivo condition. Also, the use of siRNA mimics has been reported to over-saturate cellular microRNA pathways and cause cytotoxicity (Grimm et al., 2006). (4) The full mir-302 functionality requires both sense mir-302 and its reverse mir-302* strands in the precursor, whereas current siRNA designs fail to provide the function of mir-302* (such as mir-302a*, mir-302b* and mir-302c*). As a result, an affordable and reliable method for producing and/or isolating pre-mir-302 is the key element required for developing mir-302-mediated anti-tumor/cancer therapy and its related drugs.
In sum, there remains a need for effective and safe methods of producing, isolating and utilizing mir-302 and its precursors as well as the related homologues/derivatives in drug/vaccine development and cancer therapy.